Method for treating coronary artery disease using antibody binding human protein tyrosine phosphatase beta(HPTPbeta)

ABSTRACT

Antibodies and antigen binding fragments thereof that bind to human protein tyrosine phosphatase beta (HPTPβ), and uses thereof.

This application is a Divisional Application of U.S. application Ser.No. 11/784,094, filed Apr. 5, 2007 now U.S. Pat. No. 7,973,142, whichclaims the benefit of U.S. Provisional Application No. 60/790,506, filedApr. 7, 2006 and U.S. Provisional Application No. 60/798,896, filed May9, 2006.

INCORPORATION OF SEQUENCE LISTING

The Sequence Listing filed on Jan. 25, 2010, created on Jan. 25, 2010,named 02911012220USST25.TXT, having a size in bytes of 96 kb, is herebyincorporated by reference herein in its entirety.

FIELD OF INVENTION

This invention relates to antibodies and antigen binding fragmentsthereof that bind to human protein tyrosine phosphatase beta (HPTPbeta)and uses thereof.

BACKGROUND OF THE INVENTION

Angiogenesis, the sprouting of new blood vessels from the pre-existingvasculature, plays an important role in a wide range of physiologicaland pathological processes (Nguyen, L. L. et al, Int. Rev. Cytol., 204,1-48, (2001)). Angiogenesis is a complex process, mediated bycommunication between the endothelial cells that line blood vessels andtheir surrounding environment. In the early stages of angiogenesis,tissue or tumor cells produce and secrete pro-angiogenic growth factorsin response to environmental stimuli such as hypoxia. These factorsdiffuse to nearby endothelial cells and stimulate receptors that lead tothe production and secretion of proteases that degrade the surroundingextracellular matrix. The activated endothelial cells begin to migrateand proliferate into the surrounding tissue toward the source of thesegrowth factors (Bussolino, F., Trends Biochem. Sci., 22, 251-256,(1997)). Endothelial cells then stop proliferating and differentiateinto tubular structures, which is the first step in the formation ofstable, mature blood vessels. Subsequently, periendothelial cells, suchas pericytes and smooth muscle cells, are recruited to the newly formedvessel in a further step toward vessel maturation.

Angiogenesis is regulated by a balance of naturally occurring pro- andanti-angiogenic factors. Vascular endothelial growth factor, fibroblastgrowth factor, and angiopoeitin represent a few of the many potentialpro-angiogenic growth factors. These ligands bind to their respectivereceptor tyrosine kinases on the endothelial cell surface and transducesignals that promote cell migration and proliferation. Whereas manyregulatory factors have been identified, the molecular mechanisms thatdrive this process are still not fully understood.

There are many disease states driven by persistent unregulated orimproperly regulated angiogenesis. In such disease states, unregulatedor improperly regulated angiogenesis may either cause a particulardisease or exacerbate an existing pathological condition. For example,ocular neovascularization has been implicated as the most common causeof blindness and underlies the pathology of approximately 20 eyediseases. In certain previously existing conditions, such as arthritis,newly formed capillary blood vessels invade the joints and destroycartilage. In diabetes, new capillaries formed in the retina invade thevitreous humor, causing bleeding and blindness.

Both the growth and metastasis of solid tumors may also beangiogenesis-dependent, Folkman et al., “Tumor Angiogenesis,” Chapter10, 206-32, in The Molecular Basis of Cancer, Mendelsohn et al., eds.,W. B. Saunders, (1995). It has been shown that tumors which enlarge togreater than 2 mm in diameter must obtain their own blood supply and doso by inducing the growth of new capillary blood vessels. After thesenew blood vessels become embedded in the tumor, they provide nutrientsand growth factors essential for tumor growth as well as a means fortumor cells to enter the circulation and metastasize to distant sites,such as liver, lung or bone (Weidner, New Eng. J. Med., 324, 1, 1-8(1991)). When used as drugs in tumor-bearing animals, natural inhibitorsof angiogenesis may prevent the growth of small tumors (O'Reilly et al.,Cell, 79, 315-28 (1994)). In some protocols, the application of suchinhibitors leads to tumor regression and dormancy even after cessationof treatment (O'Reilly et al., Cell, 88, 277-85 (1997)). Moreover,supplying inhibitors of angiogenesis to certain tumors may potentiatetheir response to other therapeutic regimens (see, e.g., Teischer etal., Int. J. Cancer, 57, 920-25 (1994)).

Although many disease states are driven by persistent unregulated orimproperly regulated angiogenesis, some disease states may be treated byenhancing angiogenesis. Tissue growth and repair are biologic eventswherein cellular proliferation and angiogenesis occur. Thus an importantaspect of wound repair is the revascularization of damaged tissue byangiogenesis.

Chronic, non-healing wounds are a major cause of prolonged morbidity inthe aged human population. This is especially the case in bedridden ordiabetic patients who develop severe, non-healing skin ulcers. In manyof these cases, the delay in healing is a result of inadequate bloodsupply either as a result of continuous pressure or of vascularblockage. Poor capillary circulation due to small artery atherosclerosisor venous stasis contributes to the failure to repair damaged tissue.Such tissues are often infected with microorganisms that proliferateunchallenged by the innate defense systems of the body which requirewell vascularized tissue to effectively eliminate pathogenic organisms.As a result, most therapeutic intervention centers on restoring bloodflow to ischemic tissues thereby allowing nutrients and immunologicalfactors access to the site of the wound.

Atherosclerotic lesions in large vessels may cause tissue ischemia thatcould be ameliorated by modulating blood vessel growth to the affectedtissue. For example, atherosclerotic lesions in the coronary arteriesmay cause angina and myocardial infarction that could be prevented ifone could restore blood flow by stimulating the growth of collateralarteries. Similarly, atherosclerotic lesions in the large arteries thatsupply the legs may cause ischemia in the skeletal muscle that limitsmobility and in some cases necessitates amputation, which may also beprevented by improving blood flow with angiogenic therapy.

Other diseases such as diabetes and hypertension are characterized by adecrease in the number and density of small blood vessels such asarterioles and capillaries. These small blood vessels are important forthe delivery of oxygen and nutrients. A decrease in the number anddensity of these vessels contributes to the adverse consequences ofhypertension and diabetes including claudication, ischemic ulcers,accelerated hypertension, and renal failure. These common disorders andmany other less common ailments, such as Burgers disease, could beameliorated by increasing the number and density of small blood vesselsusing angiogenic therapy.

Thus, there is a continuing need to identify regulators of angiogenesis.

In view of the foregoing, there is a need to identify biochemicaltargets in the treatment of angiogenesis mediated disorders. However,angiogenesis involves the action of multiple growth factors and theircognate receptor tyrosine kinases (RTKs), Yancopoulos et al., Nature,407,242-248, 2000). Vascular endothelial growth factor (VEGF), forexample, is important for the differentiation of endothelial cells intonascent blood vessels in the embryonic vasculature. Further, VEGFenhances blood vessel development in the adult vasculature.Administration of exogenous VEGF enhances the development of thecollateral vasculature and improves blood flow to ischemic tissues.

To date, three VEGF RTKs have been identified, VEGFR1 (FLT-1), VEGFR2(KDR), and VEGFR3 (FLT-4). Although these receptors are highlyconserved, based on biochemical characterization and biologicalactivity, each has specific and non-overlapping functions. Of the threereceptors, VEGFR2 is believed to play the predominant role in mediatingVEGF actions in the developing vasculature and during angiogenesis inadults. However, both VEGFR1 and VEGFR3 are required for normaldevelopment of the embryonic vasculature and may also be important forangiogenesis in adult tissues. Upon VEGF binding and dimerization, aconformational change in the VEGFR2 kinase domain enhances its kinaseactivity resulting in “autophosphorylation” of the other member of thepair on specific tyrosine residues. These autophosphorylation eventsserve to further enhance the kinase activity and provide anchor pointsfor the association of intracellular signaling molecules.

However, activation of a single angiogenic pathway may not be sufficientto produce persistent and functional vessels that provide adequateperfusion to ischemic tissue. These findings, together with fact thatmultiple RTKs are involved in the assembly of embryonic vasculature,indicate that biochemical targets that modulate multiple angiogenicpathways will have advantages over administration of a single growthfactor.

Protein tyrosine phosphatases (PTPs) comprise a large family of closelyrelated enzymes that dephosphorylate proteins that containphosphotyrosine residues. Recent evidence suggests that one function ofPTPs is to limit the phosphorylation and activation of RTKs. Forexample, HCPTPA, a low molecular weight protein tyrosine phosphatase,was shown to associate with VEGFR2 and negatively regulate itsactivation in cultured endothelial cells and its biological activity inangiogenesis assays, (Huang et al., Journal of Biological Chemistry,274, 38183-38185, 1999).

In addition to VEGFR2, signaling input from another RTK, Tie-2, thereceptor for the angiopoietins (Ang1 and Ang2), is also important.Deletion of either the Ang1 or Tie-2 gene in mice may result inembryonic lethality secondary to abnormalities in the developingvasculature (Yancopoulos et al., Nature, 407, 242-248, 2000). Inaddition, overexpression of Ang1 in the skin increases skin vascularityand administration of exogenous Ang1 increases blood flow to ischemicskeletal muscle (Suri et al., Science, 282, 468-471, 1998). Moreover,inhibiting the activation of Tie-2 inhibits angiogenesis and limitstumor progression in animal models of cancer, (Lin et al., J. Clin.Invest., 100, 2072-2078, 1997). In addition to its angiogenicactivities, activation of Tie-2 by exogenous administration of Ang1blocks VEGF mediated vascular leak and pro-inflammatory effects, butenhances its angiogenic effects (Thurston et al., Nature Medicine, 6,460-463, 2000). Therefore, biological targets that modulate both VEGFR2and Tie-2 signaling may yield superior proangiogenic or antiangiogenictherapies.

HPTPbeta (first described in Kruegar et al., EMBO J., 9, (1990)) hasbeen suggested for modulating the activity of angiopoietin receptor-typetyrosine kinase Tie-2, e.g., WO 00/65088). HPTPbeta is also suggestedfor regulating activities of VEGFR2, e.g., US Pat. Pub. No.2004/0077065.

It would be desirable to develop antibodies, e.g., a humanizedmonoclonal antibody, which selectively regulate the activity of HPTPbetaand thereby enhance angiogenic signaling, stimulate blood vessel growth(angiogenesis), and/or increase blood flow in ischemic tissue, or reduceangiogenic signaling, reduce blood vessel growth, and/or decrease bloodflow to the effected tissue. Herein are described antibodies andfragments thereof that bind HPTPbeta and regulate angiogenic cellsignaling, which in turn, regulates angiogenesis.

SUMMARY OF THE INVENTION

The present invention relates to antibodies that bind human proteintyrosine phosphatase beta HPTPbeta and thereby regulate angiogenic cellsignaling, which in turn, regulates angiogenesis.

In one embodiment, the invention relates to an isolated antibody orantigen-binding fragment thereof which binds to human protein tyrosinephosphatase beta, wherein said antibody or antigen-binding fragmentthereof regulates angiogenic cell signaling, which in turn, regulatesangiogenesis.

In another embodiment, the invention relates to an antibody that bindsthe N-terminal portion of human protein tyrosine phosphatase beta.

In another embodiment, the invention relates to an antibody that bindsthe first FN3 repeat of human protein tyrosine phosphatase beta.

In another embodiment, the invention relates to an antibody that bindsthe first FN3 repeat of human protein tyrosine phosphatase beta, whereinthe first FN3 repeat of human protein tyrosine phosphatase beta has thesequence as shown in SEQ ID NO: 11, or a portion thereof.

In another embodiment, the invention relates to an antibody wherein theantibody is a monoclonal antibody.

In another embodiment, the invention relates to an antibody wherein theantibody is the monoclonal antibody R15E6 (Mouse hybridoma, Balbc spleencells (B cells) deposited with American Type Culture Collection (ATCC),P.O. Box 1549, Manassas, Va. 20108 USA on 4 May 2006, assigned ATCC No.PTA-7580).

In another embodiment, the invention relates to an antibody having thesame, or substantially the same, biological characteristics of R15E6.

In another embodiment, the invention relates to an antibody, wherein theantibody or the antigen binding fragment is humanized.

In another embodiment, the invention relates to an antibody, wherein theantibody comprises antigen binding region residues from the monoclonalantibody R15E6 and is humanized.

In another embodiment, the invention relates to an antigen bindingfragment of an antibody, wherein the fragment comprises heavy and lightchain variable regions.

In another embodiment, the invention relates to an antigen bindingfragment of an antibody, wherein the antigen-binding fragment isselected from the group consisting of an Fv fragment, an Fab fragment,an Fab′ fragment, and an F(ab′)₂ fragment.

In another embodiment, the invention relates to an a method of treatingan angiogenesis regulated disorder in a subject, comprising: identifyinga subject in need of regulation of angiogenesis; and administering tothe subject an effective amount of an antibody or antigen-bindingfragment thereof which binds HPTPbeta and regulates angiogenesis.

In another embodiment, the invention relates to a method of treating anangiogenesis regulated disorder in a subject, wherein the angiogenesisregulated disorder is an angiogenesis elevated disorder, and is selectedfrom the group consisting of diabetic retinopathy, macular degeneration,cancer, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum,Paget's disease, vein occlusion, artery occlusion, carotid obstructivedisease, chronic uveitis/vitritis, mycobacterial infections, Lyme'sdisease, systemic lupus erythematosis, retinopathy of prematurity,Eales' disease, Behcet's disease, infections causing a retinitis orchoroiditis, presumed ocular histoplasmosis, Best's disease, myopia,optic pits, Stargardt's disease, pars planitis, chronic retinaldetachment, hyperviscosity syndrome, toxoplasmosis, trauma andpost-laser complications, diseases associated with rubeosis, andproliferative vitreoretinopathy.

In another embodiment, the invention relates to a method of treating anangiogenesis regulated disorder in a subject, wherein the angiogenesisregulated disorder is an angiogenesis elevated disorder, and is selectedfrom the group including but not limited to diabetic retinopathy,macular degeneration, cancer, rheumatoid arthritis, hemangiomas,Osler-Weber-Rendu disease, or hereditary hemorrhagic telangiectasia, andsolid or blood borne tumors

In another embodiment, the invention relates to a method of treating anangiogenesis regulated disorder in a subject, wherein the angiogenesisregulated disorder is an angiogenesis elevated disorder, and is selectedfrom the group consisting of inflammatory bowel diseases such as Crohn'sdisease and ulcerative colitis, psoriasis, sarcoidosis, rheumatoidarthritis, hemangiomas, Osler-Weber-Rendu disease, or hereditaryhemorrhagic telangiectasia, solid or blood borne tumors and acquiredimmune deficiency syndrome.

In another embodiment, the invention relates to a method of treating anangiogenesis regulated disorder in a subject, wherein the angiogenesisregulated disorder is an angiogenesis reduced disorder and is selectedfrom the group including but not limited to skeletal muscle ormyocardial ischemia, stroke, coronary artery disease, peripheralvascular disease, coronary artery disease, cerebrovascular disease,diabetic neuropathy and wound healing.

In another embodiment, the invention relates to a method of treating anangiogenesis regulated disorder in a subject, wherein the angiogenesisregulated disorder is an angiogenesis reduced disorder and is selectedfrom the group consisting of skeletal muscle and myocardial ischemia,stroke, coronary artery disease, peripheral vascular disease, coronaryartery disease.

In another embodiment, the invention relates to a method of treating anangiogenesis reduced disorder in a subject, wherein the angiogenesisreduced disorder is peripheral vascular disease.

In another embodiment, the invention relates to a method of treating anangiogenesis reduced disorder in a subject, wherein the angiogenesisreduced disorder is coronary artery disease.

In another embodiment, the invention relates to a pharmaceuticalcomposition, comprising: an antibody or a fragment thereof which bindsto human protein tyrosine phosphatase beta; and a pharmaceuticallyacceptable carrier.

In another embodiment, the invention relates to a pharmaceuticalcomposition, comprising: an antibody or a fragment thereof which bindsto human protein tyrosine phosphatase beta, wherein the antibody is themonoclonal antibody R15E6; and a pharmaceutically acceptable carrier.

In another embodiment, the invention relates to a pharmaceuticalcomposition, comprising: an antibody or a fragment thereof which bindsto human protein tyrosine phosphatase beta, wherein the antibody is amonoclonal antibody having the same, or substantially the same,biological characteristics of R15E6; and a pharmaceutically acceptablecarrier.

In another embodiment, the invention relates to a pharmaceuticalcomposition, comprising: an antibody or a fragment thereof which bindsto human protein tyrosine phosphatase beta, wherein the antibody or theantigen binding fragment is humanized; and a pharmaceutically acceptablecarrier.

In another embodiment, the invention relates to a pharmaceuticalcomposition, comprising: an antibody or a fragment thereof which bindsto human protein tyrosine phosphatase beta, wherein the antibodycomprises antigen binding region residues from the monoclonal antibodyR15E6 and is humanized; and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Design and production of the HPTPβ ECD protein. (Panel A)Schematic representation of the full-length HPTPβ and the HPTPβextracellular domain-6His fusion protein. (Panel B) Silver stain ofImidazole eluates from a Ni-NTA column loaded with supernatant fromHEK293 cells transfected with a vector directing the expression ofβECD-6His. A single high molecular weight band consistent with the HPTPβextracellular domain-6His protein is detected.

FIG. 2. R15E6 Recognizes Endogenous HPTPβ on Endothelial Cells. (PanelA) Endothelial cell lysates are immunoprecipitated with a controlantibody (Lane 1), with R15E6 (Lane 2) or with a mixture of anti-Tie2and anti-VEGFR2 antibodies (Lane 3). Immunoprecipitates are resolved bySDS-PAGE, transferred to a PVD membrane and probed by western blot witha mixture of R15E6, anti-Tie2 and anti-VEGFR2 antibodies. A single majorhigh molecular weight band consistent with HPTPβ is seen with R15E6(Lane 2) and not with the control antibody (Lane 1) or the mixture ofanti-Tie2 and anti-VEGFR2 (Lane 3). (Panel B) Endothelial cells aresubjected to FACS analysis with R15E6 (white peak) or a no primaryantibody control (black peak). The robust shift in fluorescenceindicates that R15E6 binds to HPTPβ on the surface of intact endothelialcells.

FIG. 3. R15E6 Enhances Tie2 Receptor Activation in HUVEC's. Tie2activation is measured in human endothelial cells as described inExample 4. R15E6 dose dependently enhances both basal and Ang1-inducedTie2 activation.

FIG. 4. R15E6 Enhances HUVEC Survival. Survival of serum starved humanendothelial cells is measured as described in Example 4. Consistent withits effects on Tie2 activation, R15E6 dose dependently enhances bothbasal and Ang1-induced endothelial cell survival (Panel A). In addition,R15E6 also dose dependently enhances VEGF and FGF-mediated endothelialcell survival (Panels B and C). A control antibody fails to enhanceendothelial cell survival (Panel D).

FIG. 5. R15E6 Enhances HUVEC Migration. Migration of human endothelialcells is measured as described in Example 4. R15E6 dose dependentlyenhances both basal and VEGF-induced endothelial cell migration.

FIG. 6. R15E6 Enhances Capillary Morphogenesis in the HUVEC/BeadSprouting Assay. Capillary morphogenesis of human endothelial cells ismeasured in the bead sprouting assay as described in Example 4. R15E6enhances both basal and VEGF-induced endothelial cell capillarymorphogenesis.

FIG. 7. Western blot analysis localizes the R15E6 binding epitope to theN-terminal FN3 repeat of the HPTPβ extracellular domain. (Panel A) Bywestern analysis, R15E6 binds to all of the C-terminal truncationmutants demonstrating that the binding epitope is located in theN-terminal 2 FN3 repeats. (Panel B) Analysis of mouse/human chimericproteins further localizes the R15E6 binding epitope to the HPTPβN-terminal FN3 repeat.

FIG. 8. MSD analysis confirms localization of the R15E6 binding epitopeto the N-terminal FN3 repeat of the HPTPβ extracellular domain. (PanelA) By MSD analysis, R15E6 binds to all of the C-terminal truncationmutants confirming that the binding epitope is located in the N-terminal2 FN3 repeats. (Panel B) Analysis of mouse/human chimeric proteinsfurther confirms the localization of the R15E6 binding epitope to theHPTPβ N-terminal FN3 repeat.

FIG. 9. MSD analysis demonstrates that the monovalent R15E6 Fab fragmentalso binds the N-terminal FN3 repeat of HPTPβ. (Panel A) Similar to theintact R15E6 antibody, the R15E6 Fab fragment binds to all of theC-terminal truncation mutants confirming that the binding epitope islocated in the N-terminal 2 FN3 repeats. (Panel B) Analysis ofmouse/human chimeric proteins further localizes the binding epitope ofthe R15E6 Fab fragment to the HPTPβ N-terminal FN3 repeat.

FIG. 10. The monovalent R15E6 Fab fragment fails to enhance Tie2activation and blocks Tie2 activation by intact R15E6.

FIG. 11. The R15E6 Fab fragment potently inhibits endothelial cellsurvival. (Panel A) Compared to a control Fab fragment, the R15E6 Fabfragment potently inhibits endothelial cell survival. (Panel B) Theinhibitory effect of the R15E6 Fab fragment is rescued by competitionwith intact R15E6.

FIG. 12. The R15E6 Fab fragment inhibits VEGF mediated endothelial cellmigration.

SEQUENCE LISTING DESCRIPTION

Each of the nucleotide and protein sequences in the sequence listing,along with the corresponding Genbank or Derwent accession number(s),where applicable, and species from which it is derived, is shown inTable I.

TABLE I SEQ ID NOs: Equivalent Sequence Nucleotide, Genbank DescriptionProtein Species Acc. No. Extracellular 1, 2 Homo Sapiens domain ofHPTPbeta with His and Gly tag Extracellular 3 Homo Sapiens X54131 domainof full- NM_002837 length HPTPbeta ½ (AA1-730, 8 4 Homo SapiensFN3's)775 aa ¼ (AA1-376, 4 5 Homo Sapiens FN3's)421 aa ⅛ (AA1-202, 2 6Homo Sapiens FN3's)247 aa Mouse full length 7 Mus musculus NM_029928ECD1632 aa First human FN3- 8 Human-mouse Mouse ½ chimera Second human 9Human-mouse FN3-Mouse ½ chimera First two human 10 Human-mouse FN3, -Mouse ½ chimera Human FN3, first 11 Homo sapines repeat

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to antibodies that bind HPTPbeta and usesthereof.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The techniques andprocedures are generally performed according to conventional methodsknown in the art and as described in various general and more specificreferences that are cited and discussed throughout the presentspecification. Unless specific definitions are provided, thenomenclature utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thoseknown and commonly used in the art. Standard techniques may be used forchemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

“Protein,” is used herein interchangeably with peptide and polypeptide.HPTPbeta is human protein tyrosine phosphatase as defined in thesequence listing. In some of the embodiments, various fragments ofHPTPbeta are used. Homologs, orthologs, fragments, variants, and mutantsof HPTPbeta protein and gene, as described below, are considered aswithin the scope of the term “HPTPbeta”.

By “fragment” is intended a portion of the nucleotide or proteinsequence. Fragments may retain the biological activity of the nativeprotein. Fragments of a nucleotide sequence are also useful ashybridization probes and primers or to regulate expression of a gene,e.g., antisense, siRNA, or micro RNA. A biologically active portion maybe prepared by isolating a portion of one of the nucleotide sequences ofthe invention, expressing the encoded portion (e.g., by recombinantexpression in vitro), and assessing the activity of the encoded protein.

One of skill in the art would also recognize that genes and proteinsfrom species other than those listed in the sequence listing,particularly vertebrate species, may be useful. Such species include,but are not limited to, mice, rats, guinea pigs, rabbits, dogs, pigs,goats, cows, monkeys, chimpanzees, sheep, hamsters, and zebrafish. Oneof skill in the art would further recognize that by using probes fromthe known species' sequences, cDNA or genomic sequences homologous tothe known sequence could be obtained from the same or alternate speciesby known cloning methods. Such homologs and orthologs are contemplatedto be useful in practicing the invention.

By “variants” are intended similar sequences. For example, conservativevariants may include those sequences that, because of the degeneracy ofthe genetic code, encode the amino acid sequence of one of thepolypeptides of the invention. Naturally occurring allelic variants, andsplice variants may be identified with the use of known techniques,e.g., with polymerase chain reaction (PCR), single nucleotidepolymorphism (SNP) analysis, and hybridization techniques. To isolateorthologs and homologs, generally stringent hybridization conditions areutilized, dictated by specific sequences, sequence length,guanine+cytosine (GC) content, and other parameters. Variant nucleotidesequences also include synthetically derived nucleotide sequences, e.g.,derived by using site-directed mutagenesis. Variants may containadditional sequences from the genomic locus alone or in combination withother sequences.

The molecules of the invention also include truncated and/or mutatedproteins wherein regions of the protein not required for ligand bindingor signaling have been deleted or modified. Similarly, they may bemutated to modify their ligand binding or signaling activities. Suchmutations may involve non-conservative mutations, deletions, oradditions of amino acids or protein domains. Variant proteins may or maynot retain biological activity. Such variants may result from, e.g.,genetic polymorphism or from human manipulation.

Fusions proteins are also contemplated herein. Using known methods, oneof skill in the art would be able to make fusion proteins of theproteins of the invention; that, while different from native form, maybe useful. For example, the fusion partner may be a signal (or leader)polypeptide sequence that co-translationally or post-translationallydirects transfer of the protein from its site of synthesis to anothersite (e.g., the yeast α-factor leader). Alternatively, it may be addedto facilitate purification or identification of the protein of theinvention (e.g., poly-His, Flag peptide, or fluorescent proteins).

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally is capable of being used in an animal toproduce antibodies capable of binding to an epitope of that antigen. Anantigen may have one or more epitopes.

The term “epitope” includes any antigenic determinant, preferably apolypeptide determinant, capable of specific binding to animmunoglobulin or a T-cell receptor. In certain embodiments, epitopedeterminants include chemically active surface groupings such as aminoacids, sugars, lipids, phosphoryl, or sulfonyl, and, in certainembodiments, may have specific three dimensional structuralcharacteristics, and/or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody. In certainembodiments, an antibody is said to specifically bind an antigen when itpreferentially recognizes its target antigen in a complex mixture ofproteins and/or macromolecules. An antibody is also said to specificallybind an antigen when it exhibits higher affinity to the antigen thanother related and/or unrelated molecules.

The term “antibody” (Ab) as used herein includes monoclonal antibodies,polyclonal antibodies, multi-specific antibodies (e.g. bispecificantibodies), single chain antibodies, e.g., antibodies from llama andcamel, antibody fragments, e.g., variable regions and/or constant regionfragments, so long as they exhibit a desired biological activity, e.g.,antigen-binding activity. The term “immunoglobulin” (Ig) is usedinterchangeably with “antibody” herein.

An “isolated antibody” is one which has been identified, and/orseparated, and/or recovered from its natural environment.

The basic four-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains (an IgM antibody consists of 5 of the basic heterotetramer unitalong with an additional polypeptide called J chain, and thereforecontain 10 antigen binding sites, while secreted IgA antibodies maypolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain). In the case of IgGs, the four-chainunit is generally about 150 kilo Daltons (kDa). Each L chain is linkedto an H chain by one covalent disulfide bond, while the two H chains arelinked to each other by one or more disulfide bonds depending on the Hchain isotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for μ and ε isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain (C_(L)) at its other end. The V_(L) is aligned with the V_(H) andthe C_(L) is aligned with the first constant domain of the heavy chain(C_(H)1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see, e.g., Basic and Clinical Immunology, 8th edition,Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton& Lange, 1994, page 71 and Chapter 6.

The L chain from any vertebrate species may be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (C_(H)),immunoglobulins may be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, ε, γ and μ, respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyminor differences in C_(H) sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

Members of the Camelidae family, e.g., llama, camel, and dromedaries,contain a unique type of antibody, that are devoid of light chains, andfurther lack the C_(H1) domain (Muyldermans, S., Rev. Mol. Biotechnol.,74, 277-302 (2001)). The variable region of these heavy chain antibodiesare termed V_(HH) or VHH, and constitute the smallest available intactantigen binding fragment (15 kDa) derived from a functionalimmunoglobulin.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines specificity of a particularantibody for its antigen. However, the variability is not evenlydistributed across the 110-amino acid span of the variable domains.Instead, the V regions consist of relatively invariant stretches calledframework regions (FR) of 15-30 amino acids separated by shorter regionsof extreme variability called “hypervariable regions” that are each 9-12amino acids long. The variable domains of native heavy and light chainseach comprise four FRs, largely adopting a β-sheet configuration,connected by three hypervariable regions, which form loops connecting,and in some cases forming part of, the β-sheet structure. Thehypervariable regions in each chain are held together in close proximityby the FRs and, with the hypervariable regions from the other chain,contribute to the formation of the antigen-binding site of antibodies.The constant domains are not involved directly in binding an antibody toan antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody dependent cellularcytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V_(L), and aroundabout 1-35 (H1), 50-65 (H2) and 95-102 (H3) in the V_(H); Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop”.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. In contrast to polyclonal antibody preparations whichinclude different antibodies directed against different epitopes, eachmonoclonal antibody is directed against a single epitope, i.e., a singleantigenic determinant. In addition to their specificity, the monoclonalantibodies are advantageous in that they may be synthesizeduncontaminated by other antibodies. The modifier “monoclonal” is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies useful in the presentinvention may be prepared by the hybridoma methodology or may be madeusing recombinant DNA methods in bacterial, eukaryotic animal or plantcells (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies”may also be isolated from phage antibody libraries, using the availabletechniques, e.g., Clackson et al., Nature, 352:624-628 (1991).

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; and Morrison et al.,Proc. Natl. Acad. Sci. USA, 81, 6851-6855 (1984)).

An “antibody fragment” comprises a portion of a multimeric antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂,dimmers and trimers of Fab conjugates, Fv, scFv, minibodies; dia-,tria-, and tetrabodies; linear antibodies (See Hudson et al, Nature Med.9, 129-134 (2003)).

“Fv” is the minimum antibody fragment which contains a complete antigenbinding site. This fragment consists of a dimer of one heavy- and onelight-chain variable region domain in tight, non-covalent association.From the folding of these two domains emanate six hypervariable loops (3loops each from the H and L chain) that contribute the amino acidresidues for antigen binding and confer antigen binding specificity tothe antibody. However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the ability torecognize and bind antigen, and are therefore included in the definitionof Fv.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding.

The terms “dia-, tria-, and tetrabodies” refer to small antibodyfragments prepared by constructing sFv fragments with short linkers(about 5-10 residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a multivalent fragment.

The term “humanized antibody” or “human antibody” refers to antibodieswhich comprise heavy and light chain variable region sequences from anon-human species (e.g., a mouse) but in which at least a portion of theVH and/or VL sequence has been altered to be more “human-like”, i.e.,more similar to human germline variable sequences. One type of humanizedantibody is a CDR-grafted antibody, in which human CDR sequences areintroduced into non-human VH and VL sequences to replace thecorresponding nonhuman CDR sequences. Means for making chimeric,CDR-grafted and humanized antibodies are known to those of ordinaryskill in the art (see, e.g., U.S. Pat. Nos. 4,816,567 and 5,225,539).One method for making human antibodies employs the use of transgenicanimals, such as a transgenic mouse. These transgenic animals contain asubstantial portion of the human antibody producing genome inserted intotheir own genome and the animal's own endogenous antibody production isrendered deficient in the production of antibodies. Methods for makingsuch transgenic animals are known in the art. Such transgenic animalsmay be made using XenoMouse™ technology or by using a “minilocus”approach. Methods for making XenoMice™ are described in U.S. Pat. Nos.6,162,963, 6,150,584, 6,114,598 and 6,075,181. Methods for makingtransgenic animals using the “minilocus” approach are described in U.S.Pat. Nos. 5,545,807, 5,545,806 and 5,625,825, and WO 93/12227.

Humanization of a non-human antibody has become routine in recent years,and is now within the knowledge of one skilled in the art. Severalcompanies provide services to make a humanized antibody, e.g., Xoma,Aries, Medarex, PDL, and Cambridge Antibody Technologies. Humanizationprotocols are extensively described in technical literature, e.g.,Kipriyanov and Le Gall, Molecular Biotechnol, Vol. 26, pp 39-60 (2004),Humana Press, Totowa, N.J.; Lo, Methods Mol. Biol., Vol. 248, pp 135-159(2004), Humana Press, Totowa, N.J.; Wu et al, J. Mol. Biol. 294, 151-162(1999).

In certain embodiments, antibodies of the present invention may beexpressed in cell lines other than hybridoma cell lines. Sequencesencoding particular antibodies may be used for transformation of asuitable mammalian host cell by known methods for introducingpolynucleotides into a host cell, including, for example packaging thepolynucleotide in a virus (or into a viral vector) and transducing ahost cell with the virus (or vector), or by transfection proceduresknown in the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040,4,740,461, and 4,959,455. The transformation procedure used may dependupon the host to be transformed. Methods for introduction ofheterologous polynucleotides into mammalian cells are known in the artand include; but are not limited to, dextran-mediated trasfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, mixing nucleic acid withpositively-charged lipids, and direct microinjection of the DNA intonuclei.

A nucleic acid molecule encoding the amino acid sequence of a heavychain constant region, a heavy chain variable region, a light chainconstant region, or a light chain variable region of an antibody, or afragment thereof in a suitable combination if desired, is/are insertedinto an appropriate expression vector using standard ligationtechniques. The antibody heavy chain or light chain constant region maybe appended to the C terminus of the appropriate variable region and isligated into an expression vector. The vector is typically selected tobe functional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery such that amplification of thegene and/or expression of the gene may occur). For a review ofexpression vectors, see Methods Enzymol. vol. 185 (Goeddel, ed.), 1990,Academic Press.

Antibodies and fragments thereof of the present invention bind HPTPbetaand regulate angiogenesis. As defined above, the term antibody is usedas to denote an antigen binding fragment. The uses of such antibodiesand antigen binding fragments are further described below.

Screening Assays Using In Vitro and In Vivo Models of Angiogenesis

Antibodies of the invention may be screened in angiogenesis assays thatare known in the art. Such assays include in vitro assays that measuresurrogates of blood vessel growth in cultured cells or formation ofvascular structures from tissue explants and in vivo assays that measureblood vessel growth directly or indirectly (Auerbach, R., et al. (2003).Clin Chem 49, 32-40, Vailhe, B., et al. (2001). Lab Invest 81, 439-452).

In Vitro Models of Angiogenesis

Most of these assays employ cultured endothelial cells or tissueexplants and measure the effect of agents on “angiogenic” cell responsesor on the formation of blood capillary-like structures. Examples of invitro angiogenesis assays include but are not limited to endothelialcell migration and proliferation, capillary tube formation, endothelialsprouting, the aortic ring explant assay and the chick aortic archassay.

In Vivo Models of Angiogenesis

In these assays agents or antibodies are administered locally orsystemically in the presence or absence of growth factors (i.e. VEGF orangiopoietin 1) and new blood vessel growth is measured by directobservation or by measuring a surrogate marker such as hemoglobincontent or a fluorescent indicator. Examples of angiogenesis include butare not limited to chick chorioallantoic membrane assay, the cornealangiogenesis assay, and the MATRIGEL™ plug assay.

Treatment of Angiogenesis Regulated Disorders

The term “regulate” is defined as in its accepted dictionary meanings.Thus, the meaning of the term “regulate” includes, but is not limitedto, up-regulate or down-regulate, to fix, to bring order or uniformity,to govern, or to direct by various means. In one aspect, an antibody maybe used in a method for the treatment of an “angiogenesis elevateddisorder” or “angiogenesis reduced disorder”. As used herein, an“angiogenesis elevated disorder” is one that involves unwanted orelevated angiogenesis in the biological manifestation of the disease,disorder, and/or condition; in the biological cascade leading to thedisorder; or as a symptom of the disorder. Similarly, the “angiogenesisreduced disorder” is one that involves wanted or reduced angiogenesis inthe biological manifestations. This “involvement” of angiogenesis in anangiogenesis elevated/reduced disorder includes, but is not limited to,the following:

(1) The angiogenesis as a “cause” of the disorder or biologicalmanifestation, whether the level of angiogenesis is elevated or reducedgenetically, by infection, by autoimmunity, trauma, biomechanicalcauses, lifestyle, or by some other causes.

(2) The angiogenesis as part of the observable manifestation of thedisease or disorder. That is, the disease or disorder is measurable interms of the increased or reduced angiogenesis. From a clinicalstandpoint, angiogenesis indicates the disease; however, angiogenesisneed not be the “hallmark” of the disease or disorder.

(3) The angiogenesis is part of the biochemical or cellular cascade thatresults in the disease or disorder. In this respect, regulation ofangiogenesis may interrupt the cascade, and may control the disease.Non-limiting examples of angiogenesis regulated disorders that may betreated by the present invention are herein described below.

Antibodies of the present invention may be used to treat diseasesassociated with retinal/choroidal neovascularization that include, butare not limited to, diabetic retinopathy, macular degeneration, cancer,sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget'sdisease, vein occlusion, artery occlusion, carotid obstructive disease,chronic uveitis/vitritis, mycobacterial infections, Lyme's disease,systemic lupus erythematosis, retinopathy of prematurity, Eales'disease, Behcet's disease, infections causing a retinitis orchoroiditis, presumed ocular histoplasmosis, Best's disease, myopia,optic pits, Stargardt's disease, pars planitis, chronic retinaldetachment, hyperviscosity syndromes, toxoplasmosis, trauma andpost-laser complications. Other diseases include, but are not limitedto, diseases associated with rubeosis (neovasculariation of the iris)and diseases caused by the abnormal proliferation of fibrovascular orfibrous tissue including all forms of proliferative vitreoretinopathy,whether or not associated with diabetes.

Antibodies of the present invention may be used to treat diseasesassociated with chronic inflammation. Diseases with symptoms of chronicinflammation include inflammatory bowel diseases such as Crohn's diseaseand ulcerative colitis, psoriasis, sarcoidosis and rheumatoid arthritis.Angiogenesis is a key element that these chronic inflammatory diseaseshave in common. The chronic inflammation depends on continuous formationof capillary sprouts to maintain an influx of inflammatory cells. Theinflux and presence of the inflammatory cells produce granulomas andthus, maintain the chronic inflammatory state. Inhibition ofangiogenesis by the compositions and methods of the present inventionwould prevent the formation of the granulomas and alleviate the disease.

Crohn's disease and ulcerative colitis are characterized by chronicinflammation and angiogenesis at various sites in the gastrointestinaltract. Crohn's disease is characterized by chronic granulomatousinflammation throughout the gastrointestinal tract consisting of newcapillary sprouts surrounded by a cylinder of inflammatory cells.Prevention of angiogenesis inhibits the formation of the sprouts andprevents the formation of granulomas. Crohn's disease occurs as achronic transmural inflammatory disease that most commonly affects thedistal ileum and colon but may also occur in any part of thegastrointestinal tract from the mouth to the anus and perianal area.Patients with Crohn's disease generally have chronic diarrhea associatedwith abdominal pain, fever, anorexia, weight loss and abdominalswelling. Ulcerative colitis is also a chronic, nonspecific,inflammatory and ulcerative disease arising in the colonic mucosa and ischaracterized by the presence of bloody diarrhea.

The inflammatory bowel diseases also show extraintestinal manifestationssuch as skin lesions. Such lesions are characterized by inflammation andangiogenesis and may occur at many sites other than the gastrointestinaltract. Antibodies of the present invention may be capable of treatingthese lesions by preventing the angiogenesis, thus reducing the influxof inflammatory cells and the lesion formation.

Sarcoidosis is another chronic inflammatory disease that ischaracterized as a multisystem granulomatous disorder. The granulomas ofthis disease may form anywhere in the body and thus the symptoms dependon the site of the granulomas and whether the disease active. Thegranulomas are created by the angiogenic capillary sprouts providing aconstant supply of inflammatory cells.

Antibodies of the present invention may also treat the chronicinflammatory conditions associated with psoriasis. Psoriasis, a skindisease, is another chronic and recurrent disease that is characterizedby papules and plaques of various sizes. Prevention of the formation ofthe new blood vessels necessary to maintain the characteristic lesionsleads to relief from the symptoms.

Rheumatoid arthritis is a chronic inflammatory disease characterized bynonspecific inflammation of the peripheral joints. It is believed thatthe blood vessels in the synovial lining of the joints undergoangiogenesis. In addition to forming new vascular networks, theendothelial cells release factors and reactive oxygen species that leadto pannus growth and cartilage destruction. The factors involved inangiogenesis may actively contribute to, and help maintain, thechronically inflamed state of rheumatoid arthritis. Other diseases thatmay be treated according to the present invention are hemangiomas,Osler-Weber-Rendu disease, or hereditary hemorrhagic telangiectasia,solid or blood borne tumors and acquired immune deficiency syndrome.

Antibodies of the present invention may also be used to treat an“angiogenesis reduced disorder”. As used herein, an “angiogenesisreduced disorder” is one that angiogenesis would be consideredbeneficial to treat a disease, disorder, and/or condition. The disorderis one characterized by tissue that is suffering from or at risk ofsuffering from ischemic damage, infection, and/or poor healing, whichresults when the tissue is deprived of an adequate supply of oxygenatedblood due to inadequate circulation. As used herein, “tissue” is used inthe broadest sense, to include, but not limited to, the following:cardiac tissue, such as myocardium and cardiac ventricles; erectiletissue; skeletal muscle; neurological tissue, such as from thecerebellum; internal organs, such as the brain, heart, pancreas, liver,spleen, and lung; or generalized area of the body such as entire limbs,a foot, or distal appendages such as fingers or toes.

Methods of Vascularizing Ischemic Tissue

In one aspect, antibodies may be used in a method of vascularizingischemic tissue. As used herein, “ischemic tissue,” means tissue that isdeprived of adequate blood flow. Examples of ischemic tissue include,but are not limited to, tissue that lack adequate blood supply resultingfrom myocardial and cerebral infarctions, mesenteric or limb ischemia,or the result of a vascular occlusion or stenosis. In one example, theinterruption of the supply of oxygenated blood may be caused by avascular occlusion. Such vascular occlusion may be caused byarteriosclerosis, trauma, surgical procedures, disease, and/or otheretiologies. Standard routine techniques are available to determine if atissue is at risk of suffering ischemic damage from undesirable vascularocclusion. For example, in myocardial disease these methods include avariety of imaging techniques (e.g., radiotracer methodologies, x-ray,and MRI) and physiological tests. Therefore, induction of angiogenesisis an effective means of preventing or attenuating ischemia in tissuesaffected by or at risk of being affected by a vascular occlusion.Further, the treatment of skeletal muscle and myocardial ischemia,stroke, coronary artery disease, peripheral vascular disease, coronaryartery disease is fully contemplated.

A person skilled in the art of using standard techniques may measure thevascularization of tissue. Non-limiting examples of measuringvascularization in a subject include SPECT (single photon emissioncomputed tomography); PET (positron emission tomography); MRI (magneticresonance imaging); and combination thereof, by measuring blood flow totissue before and after treatment. Angiography may be used as anassessment of macroscopic vascularity. Histologic evaluation may be usedto quantify vascularity at the small vessel level. These and othertechniques are discussed in Simons, et al., “Clinical trials in coronaryangiogenesis,” Circulation, 102, 73-86 (2000).

Methods of Repairing Tissue

In one aspect, antibodies may be used in a method of repairing tissue.As used herein, “repairing tissue” means promoting tissue repair,regeneration, growth, and/or maintenance including, but not limited to,wound repair or tissue engineering. One skilled in the art appreciatesthat new blood vessel formation is required for tissue repair. In turn,tissue may be damaged by, including, but not limited to, traumaticinjuries or conditions including arthritis, osteoporosis and otherskeletal disorders, and burns. Tissue may also be damaged by injuriesdue to surgical procedures, irradiation, laceration, toxic chemicals,viral infection or bacterial infections, or burns. Tissue in need ofrepair also includes non-healing wounds. Examples of non-healing woundsinclude non-healing skin ulcers resulting from diabetic pathology; orfractures that do not heal readily.

Antibodies may also be used in tissue repair in the context of guidedtissue regeneration (GTR) procedures. Such procedures are currently usedby those skilled in the arts to accelerate wound healing followinginvasive surgical procedures.

Antibodies may be used in a method of promoting tissue repaircharacterized by enhanced tissue growth during the process of tissueengineering. As used herein, “tissue engineering” is defined as thecreation, design, and fabrication of biological prosthetic devices, incombination with synthetic or natural materials, for the augmentation orreplacement of body tissues and organs. Thus, the present methods may beused to augment the design and growth of human tissues outside the bodyfor later implantation in the repair or replacement of diseased tissues.For example, antibodies may be useful in promoting the growth of skingraft replacements that are used as a therapy in the treatment of burns.

In another aspect of tissue engineering, antibodies of the presentinvention may be included in cell-containing or cell-free devices thatinduce the regeneration of functional human tissues when implanted at asite that requires regeneration. As previously discussed,biomaterial-guided tissue regeneration may be used to promote boneregrowth in, for example, periodontal disease. Thus, antibodies may beused to promote the growth of reconstituted tissues assembled intothree-dimensional configurations at the site of a wound or other tissuein need of such repair.

In another aspect of tissue engineering, antibodies may be included inexternal or internal devices containing human tissues designed toreplace the function of diseased internal tissues. This approachinvolves isolating cells from the body, placing them with structuralmatrices, and implanting the new system inside the body or using thesystem outside the body. For example, antibodies may be included in acell-lined vascular graft to promote the growth of the cells containedin the graft. It is envisioned that the methods of the invention may beused to augment tissue repair, regeneration and engineering in productssuch as cartilage and bone, central nervous system tissues, muscle,liver, and pancreatic islet (insulin-producing) cells.

Pharmaceutical Formulations and Methods for Use

The antibodies of the invention may be administered to individuals totreat or to prevent diseases or disorders that are regulated by genesand proteins of the invention. The term “treatment” is used herein tomean that administration of a compound of the present inventionmitigates a disease or a disorder in a host. Thus, the term “treatment”includes, preventing a disorder from occurring in a host, particularlywhen the host is predisposed to acquiring the disease, but has not yetbeen diagnosed with the disease; inhibiting the disorder; and/oralleviating or reversing the disorder. Insofar as the methods of thepresent invention are directed to preventing disorders, it is understoodthat the term “prevent” does not require that the disease state becompletely thwarted. (See Webster's Ninth Collegiate Dictionary.)Rather, as used herein, the term preventing refers to the ability of theskilled artisan to identify a population that is susceptible todisorders, such that administration of the compounds of the presentinvention may occur prior to onset of a disease. The term does not implythat the disease state be completely avoided. The compounds identifiedby the screening methods of the present invention may be administered inconjunction with other compounds.

Safety and therapeutic efficacy of compounds identified may bedetermined by standard procedures using in vitro or in vivotechnologies. Compounds that exhibit large therapeutic indices may bepreferred, although compounds with lower therapeutic indices may beuseful if the level of side effects is acceptable. The data obtainedfrom the in vitro and in vivo toxicological and pharmacologicaltechniques may be used to formulate the range of doses.

Effectiveness of a compound may further be assessed either in animalmodels or in clinical trials of patients with unregulated or improperlyregulated angiogenesis.

As used herein, “pharmaceutically acceptable carrier” is intended toinclude all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is known in theart. Except insofar as any conventional media or agent is incompatiblewith the active compound, such media may be used in the compositions ofthe invention. Supplementary active compounds may also be incorporatedinto the compositions. A pharmaceutical composition of the invention isformulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g.,intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application may include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerin, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH may be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation may be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water-soluble), or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CREMOPHOREL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier may be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity may be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms may be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions maybe brought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients. In the caseof sterile powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Systemic administration may also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration may beaccomplished using nasal sprays or suppositories. For transdermaladministration, the active compounds are formulated into ointments,salves, gels, or creams as generally known in the art.

The compounds may also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers may be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials may also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) may also be used as pharmaceuticallyacceptable carriers. These may be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. “Dosage unit form” as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated, each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms are dictated by and are directly dependent onthe unique characteristics of the active compound and the particulartherapeutic effect to be achieved, and the limitations inherent in theart of compounding such an active compound for the treatment ofindividuals.

EXAMPLES Example 1 Production of the HPTPβ Extracellular Domain Protein

Methods: Full length HPTPβ is cloned from a human placental libraryaccording to the manufacturer's (Origene) instructions. The clone isidentical to a previously reported cDNA clone (Genbank accession#X54131) except it is missing FNIII repeat #5. A cDNA encoding theentire soluble extracellular domain (ECD) of HPTPβ is cloned by PCR fromthe full length cDNA (see sequence below) coding for AA 1-1534 with anadded c-terminal His-His-His-His-His-His-Gly (6His-Gly) (SEQ ID NO: 1).The resulting cDNA is cloned into mammalian expression vectors fortransient (pShuttle-CMV) or stable (pcDNA3.1(−)) expression in HEK293cells. To obtain purified HPTPβ ECD (βECD), HEK293 cells transfectedwith a βECD expression vector are incubated in OptiMEM-serum free(Gibco) for 24 hours under normal growth conditions. The conditionedmedia is then recovered, centrifuged to remove debris (1000 rpm×5minutes), and 1 mL of washed Ni-NTA agarose (Qiagen) (500 μL packedmaterial) is added to each 10 mL of cleared media and allowed to rockovernight at 4° C. On the following day, the mixture is loaded into acolumn and washed with 20 bed volumes of 50 mM NaH2PO4, 300 mM NaCl, 20mM Imidazole, pH 8. The purified HPTPβ extracellular domain protein (SEQID NO: 2) is then eluted in six fractions with 200 μL/elution in 50 mMNaH2PO4, 300 mM NaCl, 250 mM Imidazole, pH 8. Fractions are analyzed forprotein content using reducing-denaturing SDS-polyacrylimide gelelectrophoresis and detected by silver stain (Invitrogen) and confirmedby mass spectrometry.

Results: To develop an antibody to the extracellular domain of HPTPβ,expression vectors directing the expression of a 6-His tagged HPTPβextracellular domain protein (FIG. 1, Panel A) are developed.Subsequently, the 6-His tagged HPTPβ extracellular domain protein ispurified to near homogeneity (FIG. 1, Panel B) from the conditionedmedia of HEK293 cells transfected with the expression vector.

Example 2 Generation of Monoclonal Antibodies to HPTPβ ExtracellularDomain

Methods: For production of the HPTPβ extracellular domain immunogen, thepurified HPTPβ extracellular domain-6His protein is conjugated toporcine thyroglobulin (Sigma) using EDC coupling chemistry (Hockfield,S. et al, (1993) Cold Spring Habor Laboratory Press. Volume 1 pp.111-201, Immunocytochemistry). The resulting HPTPβ extracellulardomain-thyroglobulin conjugate is dialyzed against PBS, pH 7.4. AdultBalb/c mice are then immunized subcutaneously with the conjugate(100-200 μg) and complete Freund's adjuvant in a 1:1 mixture. After 2-3weeks, the mice are injected intraperitoneally or subcutaneously withincomplete Freund's adjuvant and the conjugate in a 1:1 mixture. Theinjection is repeated at 4-6 weeks. Sera are collected from mice 7 dayspost-third-injection and assayed for immunoreactivity to HPTPβextracellular domain antigen by ELISA and western blotting. Mice thatdisplay a good response to the antigen are boosted by a singleintra-spleen injection with 50 μl of purified HPTPβ extracellular domainprotein mixed 1:1 with Alum hydroxide using a 31 gauge extra long needle(Goding, J. W., (1996) Monoclonal Antibodies: Principles and Practices.Third Edition, Academic Press Limited. p. 145). Briefly, mice areanesthetized with 2.5% avertin, and a 1 centimeter incision is createdon the skin and left oblique body wall. The antigen mixture isadministered by inserting the needle from the posterior portion to theanterior portion of the spleen in a longitudinal injection. The bodywall is sutured and the skin is sealed with two small metal clips. Miceare monitored for safe recovery. Four days after surgery the mousespleen is removed and single cell suspensions are made for fusion withmouse myeloma cells for the creation of hybridoma cell lines (Spitz, M.,(1986) Methods In Enzymology, Volume 121. Eds. John J, Lagone and HelenVan Vunakis. PP. 33-41 (Academic Press, New York, N.Y.)). Resultinghybridomas are cultured in Dulbeccos modified media (Gibco) supplementedwith 15% fetal calf serum (Hyclone) and hypoxathine, aminopterin andthymidine.

Screening for positive hybridomas begins 8 days after the fusion andcontinues for 15 days. Hybridomas producing anti-HPTPβ extracellulardomain antibodies are identified by ELISA on two sets of 96-well plates:one coated with the histidine tagged-HPTPβ extracellular domain andanother one coated with a histidine-tagged bacterial MurA protein as anegative control. The secondary antibody is a donkey anti-mouse IgGlabeled with horseradish peroxidase (HRP) (Jackson Immunoresearch)Immunoreactivity is monitored in wells using color development initiatedby ABTS tablets dissolved in TBS buffer, pH 7.5. The individual HRPreaction mixtures are terminated by adding 100 microliters of 1% SDS andreading absorbance at 405 nm with a spectrophotometer. Hybridomasproducing antibodies that interact with HPTPβ extracellular domain-6His,and not with the murA-6His protein are used for further analysis.Limiting dilutions (0.8 cells per well) are performed twice on positiveclones in 96 well plates, with clonality defined as having greater than99% of the wells with positive reactivity. Isotypes of antibodies aredetermined using the iso-strip technology (Roche). To obtain purifiedantibody for further evaluation, tissue culture supernatants areaffinity purified using a protein A or protein G columns.

Results: Five monoclonal antibodies immunoreactive to HPTPβextracellular domain protein are isolated and given the followingnomenclature, R15E6, R12A7, R3A2, R11C3, R15G2 and R5A8.

The monoclonal antibody R15E6 is deposited with American Type CultureCollection (ATCC), P.O. Box 1549, Manassas, Va. 20108 USA on 4 May 2006.

Example 3 R15E6 Binds to Endogenous HPTPβ on Human Endothelial Cells

A. R15E6 Binds Endogenous HPTPβ as Demonstrated by Immunoprecipitationand Western Blot.

Materials: Human umbilical vein endothelial cells (HUVECs), EGM media,and trypsin neutralizing solution from Cambrex; OPTIMEM I (Gibco),bovine serum albumin (BSA; Santa Cruz), phosphate buffered saline (PBS;Gibco), Growth Factors including Angiopoietin 1 (Ang1), vascularendothelial growth factor (VEGF) and fibroblast growth factor (FGF) (R&DSystems), Tie2 monoclonal antibody (Duke University/P&GP), VEGF receptor2 (VEGFR2) polyclonal antibody (Whitaker et. al), protein A/G agarose(Santa Cruz), Tris-Glycine pre-cast gel electrophoresis/transfer system(6-8%) (Invitrogen), PVDF membranes (Invitrogen), lysis buffer (20 mmTris-HCl, 137 mm NaCl, 10% glycerol, 1% triton-X-100, 2 mM EDTA, 1 mMNaOV, 1 mM NaF, 1 mM PMSF, 1 μg/ml leupeptin, 1 μg/ml pepstatin).

Method: HUVECs are pre-treated for 30 min with antibody (in OPTIMEM) orOPTIMEM I alone. After removal of pre-treatment, cells are treated withAng1 (100 ng/ml) for 6 minutes in PBS+0.2% BSA and lysed in lysisbuffer. Lysates are run directly on a Tris-Glycine gel orimmunoprecipitated with 2-5 μg/ml Tie-2 antibody or 10 μg/ml R15E6antibody and protein A/G agarose Immunoprecipitated samples are rinsed1× with lysis buffer and boiled for 5 min in 1× sample buffer. Samplesare resolved on a Tris-Glycine gel, transferred to a PVDF membrane, anddetected by western blot using the indicated antibodies (pTYR Ab (PY99,Santa Cruz), Tie-2, VEGFR2 and/or R15E6).

Results: By IP/western blotting, R15E6 recognizes a major, highmolecular weight band consistent with the size of HPTPβ (FIG. 2, PanelA, Lane 2). The less intense, lower molecular weight bands likelyrepresent less glycosylated precursor forms of HPTPβ. Animmunoprecipitation (IP) with control, non-immune IgG shows no bands inthe molecular weight range of HPTPβ (FIG. 2, Panel A, Lane 1), and acombined Tie2/VEGFR2 IP shows bands of the expected molecular weight(FIG. 2, Panel A, Lane 3). This result demonstrates that R15E6recognizes and is specific for HPTPβ.

B. R15E6 Binds Endogenous HPTPβ as Demonstrated by FACS Analysis

Materials: HUVECs, EGM media, and trypsin neutralizing solution fromCambrex; Secondary Alexfluor 488-tagged antibody from Molecular Probes;Hanks balanced salt solution (Gibco); FACSCAN flow cytometer andCellQuest software from Becton Dickenson.

Method: HUVECs are trypsinized, treated with trypsin neutralizingsolution and rinsed with HBSS. R15E6 antibody (0.6 μg) is added to250,000 cells in 50 n1 of HBSS and incubated on ice for 20 minutes.Cells are rinsed with 1 ml HBSS followed by adding 2 μg offluorescent-conjugated secondary antibody for 20 minutes on ice. Cellsare rinsed and resuspended in 1 ml HBSS then analyzed on the FACSCANflow cytometer with CellQuest software. Control cells are treated withfluorescent-conjugated secondary antibody only.

Results: By FACS analysis, intact HUVECs, R15E6 causes a robust shift(>90% of cells) in the fluorescence signal compared to the secondaryantibody alone (FIG. 2, Panel B). This result indicates that R15E6 bindsto endogenous HPTPβ presented on the surface of intact endothelialcells.

Example 4 R15E6 Enhances Tie2 Activation, and Promotes MultipleAngiogenic Responses (Endothelial Cell Survival, Migration and CapillaryMorphogenesis)

A. R15E6 Enhances Tie2 Phosphorylation in the Absence and Presence ofthe Angiopoietin 1 (Ang1), the Tie2 Ligand.

Methods: HUVECs are cultured in serum free media as described above inthe presence or absence of various concentrations of R15E6 and with orwithout added Ang1. Lysates are prepared, immunoprecipitated with a Tie2antibody, resolved by polyacrylamide gel electrophoresis and transferredto a PVDF membrane. Membrane-bound immunoprecipitated proteins are thenserially western blotted with an antiphosphotyrosine antibody toquantify Tie2 phosphorylation followed by a Tie2 antibody to quantifytotal Tie2. Tie2 phosphorylation is expressed as the ratio of theantiphosphotyrosine signal over the total Tie2 signal.

Results: R15E6 enhances Tie2 phosphorylation both in the absence andpresence of Ang1 (FIG. 3). This result indicates that binding of R15E6to HPTPβ on the surface of endothelial cells modulates its biologicalfunction resulting in enhanced activation of Tie2 in the absence orpresence of ligand.

B. R15E6 Enhances Endothelial Cell Survival in the Absence and in thePresence of Endothelial Growth Factors.

Materials: HUVECs, EGM media, and trypsin neutralizing solution fromCambrex; DMEM (Cell Gro), Delipidized BSA (BD Falcon), Cell Titer GloATP Assay (Promega), Growth Factors (Ang1, VEGF 165, and FGF) (R&DSystems), Victor V Multilabel plate reader (Perkin Elmer Wallac).

Method: HUVECs are plated at 10,000 cells/well, serum starved inDMEM/0.2% BSA and treated for 72 h in the presence or absence of growthfactor (Ang1, VEGF, or FGF), with or without various concentrations ofR15E6 antibody. After 72 hours, the cells are rinsed with DMEM andsurviving cells are quantified by measuring ATP levels using the CellTiter Glo Luminescence Assay according to manufacturer's instructions(Promega).

Results: Consistent with the results of the Tie2 activation assay, R15E6enhances endothelial cell survival in the absence of added growth factorat concentrations between 0.5 and 5 nM (FIG. 4, Panel A). Similarly,R15E6 enhances Ang1 mediated endothelial cell survival (FIG. 4, Panel A)as well as cell survival mediated by VEGF and FGF (FIG. 4, Panels B andC). No enhanced survival is seen with a control monoclonal antibody(FIG. 4, Panel D). These results demonstrate that R15E6 binding to HPTPβon the endothelial cell surface enhances baseline endothelial cellsurvival as well as cell survival mediated by multiple angiogenicpathways (Ang1, VEGF, and FGF).

C. R15E6 Enhances Endothelial Cell Migration in the Absence and in thePresence of VEGF.

Materials: HUVECs, EGM media, and trypsin neutralizing solution fromCambrex; EBM-phenol red free (PRF-EBM, Cambrex), Delipidized BSA (BDFalcon), BD Falcon Biocoat Endothelial Cell Migration system (BDFalcon), Calcein AM (Molecular Probes); Growth Factors (VEGF 165) (R&DSystems), Victor V Multilabel plate reader (Perkin Elmer Wallac).

Method: HUVECs are resuspended in PRF-EBM+0.1% BSA and plated at 50,000cells/transwell (BD Bioscience, 3 μm pore size). Growth Factor/R15E6 isplaced in the bottom well of the transwell chamber and incubated 4-22 h.Cells migrating through the membrane are detected by labeling with 4μg/ml Calcein AM for 90′. Fluorescence is measured using a Victor Vinstrument (485/535).

Results: Consistent with the results in the survival study, R15E6enhances both baseline and VEGF-mediated endothelial cell migration(FIG. 5).

D. R15E6 Enhances Endothelial Cell Sprouting and Capillary Morphogenesisin the Absence and in the Presence of Endothelial Growth Factors.

Materials: HUVECs and EGM media from Cambrex; Cytodex beads and type Icollagen from Sigma; Dulbecco's PBS and M199 media from Gibco; VEGF fromR&D.

Method: HUVECs passage 4 (2×10⁶ cells) are cultured with 5 mg of Cytodexbeads in 10 ml of EGM in 100 mm non-tissue culture treatedbacteriological dishes for 48 hours with occasional swirling. Cellcoated beads are transferred to a 50 ml conical tube and resuspended in380 μl D-PBS. Collagen gels are prepared by adding 71.4 μl of cellcoated beads to 2.8 ml of a matrix solution consisting of 3 mg/mlcollagen in M199 media supplemented with 0.005 N NaOH, 20 mM HEPES, and26 mM NaHCO₃. Three hundred and fifty microliters of the beads aredispensed into a well on a 24 well tissue culture plate and the matrixis allowed to solidify for 1 hour at 37° C./5% CO₂. One ml of EGM mediumwith or without VEGF (10 ng/ml) or R15E6 (7.5 μg/ml) is added per welland returned to the incubator. After 48 hours, a blinded observervisualizes the sprouts with a phase contrast inverted microscope andobserves 50 beads per well, in triplicate wells, for the presence ofendothelial cell sprouts. Results are expressed as the number of sproutsper bead.

Results: Consistent with the results in the other assays, R15E6 alsoenhances baseline and VEGF mediated capillary morphogenesis in theendothelial bead sprouting assay (FIG. 6).

Example 5 The Binding Epitope for R15E6 is in the N-Terminal FN3 Repeatof the Human HPTPβ Extracellular Domain

A. Western Blot Analysis of Recombinant c-Terminal Truncation Mutantsand Mouse/Human Chimeric Proteins Show that the R15E6 Binding Epitope isin the N-Terminal FN3 Repeat of the HPTPβ Extracellular Domain.

Methods: HEK293 cells are transfected with expression vectors encodingthe indicated HPTPβ truncation mutant or mouse/human chimera.Transfected cells are then incubated in OptiMEM for an additional 24hours after which conditioned media containing the indicated HPTPβextracellular domain is harvested and either stored for future use orused immediately for western blot or ECL (see below) studies. Forwestern blot analysis, 20 μl of conditioned media containing theindicated HPTPβ protein or no recombinant protein (Mock, empty vectortransfected) is resolved by PAGE, transferred to a PVDF membrane andprobed with R15E6.

Results: By western blot analysis, R15E6 binds all of the HPTPβC-terminal deletion mutants (FIG. 7A) indicating that the bindingepitope is within the first two N-terminal FN3 repeats. R15E6 fails tobind murine HPTPβ (SEQ ID NO: 7) extracellular domain demonstratingspecificity for the human protein (FIG. 7B lane 6 vs. lane 2). Replacingthe murine 1st or 1st and 2nd N-terminal FN3 repeats with the humansequences restored R15E6 binding (FIG. 7B lanes 3 and 5). Conversely,replacing only the murine 2nd FN3 repeat with the human sequence failsto restore binding (FIG. 7B lane 4). Taken together, these findingslocalize the binding epitope of R15E6 to the N-terminal FN3 repeat (˜100amino acids) of human HPTPβ.

B. ECL (Electrochemiluminescent) Analysis of -Terminal TruncationMutants and Mouse/Human Chimeric Proteins Confirms that the R15E6Binding Epitope is in the N-Terminal FN3 Repeat of the HPTPβExtracellular Domain.

Methods: Supernatants containing the indicated HPTPβ protein are coatedon a 96 well High bind MSD (Meso Scale Discovery) plate, allowed to dry,and blocked with 3% BSA for 1 h. The protein is then incubated with theR15E6 monoclonal antibody or the R15E6 Fab fragment (10 nM or 1.5 μg/ml)for 1 h, rinsed, and incubated with a goat anti-mouse antibody with anMSD-Tag label (10 nM) for 1 h. The excess antibody is rinsed off and MSDread buffer is added. Light emission is measured using the Sector 2400reader (MSD). MSD utilizes electrochemiluminescent detection to detectbinding events on patterned arrays. Meso Scale Discovery's technologyuses proprietary MULTI-ARRAY™ and MULTI-SPOT™ microplates withelectrodes integrated into the bottom of the plate. MSD's electrodes aremade from carbon and biological reagents may be attached to the carbonsimply by passive adsorption and retain a high level of biologicalactivity. MSD assays use electrochemiluminescent labels forultra-sensitive detection. These electrochemiluminescent labels emitlight when electrochemically stimulated. The detection process isinitiated at the built in electrodes located in the bottom of MSD'smicroplates and only labels near the electrode are excited and lightdetected at 620 nm.

Results: Consistent with the western blot studies, R15E6 binds to all ofthe HPTPβ C-terminal truncation proteins by MSD analysis (FIG. 8A). Alsoconsistent with the western blot analysis, R15E6 fails to bind themurine HPTPβ extracellular domain but binding is restored by replacingthe murine N-terminal FN3 repeat with the human N-terminal FN3 domain(FIG. 8B). These data confirm that the binding epitope of R15E6 is inthe N-terminal FN3 repeat of human HPTPβ. As expected, the bindingepitope of the monovalent R15E6 Fab fragment could also be mapped to theN-terminal most FN3 repeat of human HPTPβ (FIG. 9).

Example 6 A Monovalent R15E6 Fab Fragment Blocks R15E6 Mediated Tie2Activation and Inhibits Endothelial Cell Survival and Migration

Methods: Tie2 activation and endothelial cell survival and migrationassays are performed as described above in example 4. Monovalent R15E6Fab fragments are prepared as previously described. Purified R15E6 isdialyzed in 0.1M Tris-HCL, pH 8.0, containing 2 mM EDTA and 1 mMdithiothreitol. Papain (Pierce) at 1-2 mg/ml is activated in theaforementioned buffer for 15 minutes at 37° C. R15E6 at 10 mg/ml isincubated with papain in the same buffer using an enzyme:substrate ratioof 1:100, for 1 h at 37° C. The digestion is terminated by the additionof iodoacetamide (final concentration 20 mM, and held on ice for 1 h,protected from light. The papain digested material is dialyzed overnightagainst phosphate-buffered saline, to remove iodoacetamide. The extentof digestion is monitored by SDS-PAGE with the disappearance of thegamma heavy chain (MW 55,000 kDa) and the appearance of the Fc fragmentof gamma (MW 27,000 kDa) and light chains (MW 22,000-25,000 kDa).

Results: Unlike the intact R15E6 antibody, the Fab fragments fails toenhance Tie2 activation (FIG. 10). Moreover, in the presence of excessFab fragment, R15E6-mediated Tie2 activation is blocked (FIG. 10).Surprisingly, the R15E6 Fab fragment markedly inhibits endothelial cellsurvival compared to a control Fab (FIG. 11A) and this effect could berescued by the addition of intact R15E6 (FIG. 11B). Consistent with thenegative effect on endothelial survival, the R15E6 Fab also blocks VEGFmediated endothelial cell migration (FIG. 12). These findingsdemonstrate that the intact, dimeric R15E6 is required for theenhancement of angiogenic signaling and that monomeric R15E6 blocksthese actions and actually has a negative effect on angiogenicendothelial cell responses.

Except as otherwise noted, all amounts including quantities,percentages, portions, and proportions, are understood to be modified bythe word “about”, and amounts are not intended to indicate significantdigits.

Except as otherwise noted, the articles “a”, “an”, and “the” mean “oneor more”.

All documents cited are, in relevant part, incorporated herein byreference; the citation of any document is not to be construed as anadmission that it is prior art with respect to the present invention. Tothe extent that any meaning or definition of a term in this writtendocument conflicts with any meaning or definition of the term in adocument incorporated by reference, the meaning or definition assignedto the term in this written document shall govern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications may be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed is:
 1. A method of treating skeletal muscle ormyocardial ischemia, stroke, coronary artery disease, or peripheralvascular disease, comprising administering to the subject an effectiveamount of an antibody or antigen-binding fragment thereof which bindsHPTPbeta and regulates angiogenesis.
 2. A method according to claim 1,wherein the antibody is the monoclonal antibody R15E6 produced byhybridoma cell line ATCC No. PTA-7580 which binds to human proteintyrosine phosphatase beta (HPTPβ).
 3. A method according to claim 1,wherein the antigen-binding fragment is selected from the groupconsisting of an Fv fragment, an Fab fragment, an Fab′ fragment, and anF(ab′)₂ fragment.
 4. A method of enhancing Tie-2 signaling in a subjecthaving skeletal muscle or myocardial ischemia, stroke, coronary arterydisease, or peripheral vascular disease, comprising administering to thesubject an effective amount of an antibody or antigen-binding fragmentthereof which binds HPTPbeta and regulates angiogenesis.
 5. A methodaccording to claim 4, wherein the antibody is the monoclonal antibodyR15E6 produced by hybridoma cell line ATCC No. PTA-7580 which binds tohuman protein tyrosine phosphatase beta (HPTPβ).
 6. A method accordingto claim 4, wherein the antigen-binding fragment is selected from thegroup consisting of an Fv fragment, an Fab fragment, an Fab′ fragment,and an F(ab′)₂ fragment.
 7. A method for promoting endothelial cellmigration in a subject having skeletal muscle or myocardial ischemia,stroke, coronary artery disease, or peripheral vascular disease,comprising administering to the subject an effective amount of anantibody or antigen-binding fragment thereof which binds HPTPbeta andregulates angiogenesis.
 8. A method according to claim 7, wherein theantibody is the monoclonal antibody R15E6 produced by hybridoma cellline ATCC No. PTA-7580 which binds to human protein tyrosine phosphatasebeta (HPTPβ).
 9. A method according to claim 7, wherein theantigen-binding fragment is selected from the group consisting of an Fvfragment, an Fab fragment, an Fab′ fragment, and an F(ab′)₂ fragment.10. A method for promoting endothelial cell survival in a subject havingskeletal muscle or myocardial ischemia, stroke, coronary artery disease,or peripheral vascular disease, comprising administering to the subjectan effective amount of an antibody or antigen-binding fragment thereofwhich binds HPTPbeta and regulates angiogenesis.
 11. A method accordingto claim 10, wherein the antibody is the monoclonal antibody R15E6produced by hybridoma cell line ATCC No. PTA-7580 which binds to humanprotein tyrosine phosphatase beta (HPTPβ).
 12. A method according toclaim 10, wherein the antigen-binding fragment is selected from thegroup consisting of an Fv fragment, an Fab fragment, an Fab′ fragment,and an F(ab′)₂ fragment.
 13. A method for promoting blood vesselmorphogenesis and blood vessel stabilization in a subject havingskeletal muscle or myocardial ischemia, stroke, coronary artery disease,or peripheral vascular disease, comprising administering to the subjectan effective amount of an antibody or antigen-binding fragment thereofwhich binds HPTPbeta and regulates angiogenesis.
 14. A method accordingto claim 13, wherein the antibody is the monoclonal antibody R15E6produced by hybridoma cell line ATCC No. PTA-7580 which binds to humanprotein tyrosine phosphatase beta (HPTPβ).
 15. A method according toclaim 13, wherein the antigen-binding fragment is selected from thegroup consisting of an Fv fragment, an Fab fragment, an Fab′ fragment,and an F(ab′)₂ fragment.